Enzymatic Hydroxylation of Aliphatic Hydrocarbon

ABSTRACT

The invention relates to enzymatic methods for hydroxylation in position 2 or 3 of substituted or unsubstituted, linear or branched aliphatic hydrocarbons.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/637,716filed on Sep. 27, 2012, now pending, which is a 35 U.S.C. 371 nationalapplication of PCT/EP2011/054761 filed Mar. 28, 2011, which claimspriority or the benefit under 35 U.S.C. 119 of European application nos.10158092.6 and 10158093.4 filed on Mar. 28, 2010 and Mar. 28, 2010 andU.S. provisional application No. 61/318,582 filed on Mar. 29, 2010. Thecontent of each application is fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of polypeptides havingperoxygenase activity for site specific hydroxylation of aliphatichydrocarbons.

2. Background

A peroxygenase denoted AaP from the agaric basidiomycete strain Agrocybeaegerita (strain TM-A1) was found to oxidize aryl alcohols andaldehydes. The AaP peroxygenase was purified from A. aegerita TM A1 byseveral steps of ion chromatography and SDS-PAGE, the molecular weightwas determined and the N-terminal 14 amino acid sequence was determinedafter 2-D electrophoresis but the encoding gene was not isolated(Ullrich et al., 2004, Appl. Env. Microbiol. 70(8): 4575-4581).

WO 2006/034702 discloses methods for the enzymatic hydroxylation ofnon-activated hydrocarbons, such as, naphtalene, toluol and cyclohexane,using the AaP peroxygenase enzyme of Agrocybe aegerita TM A1. This isalso described in Ullrich and Hofrichter, 2005, FEBS Letters 579:6247-6250.

WO 2008/119780 discloses eight different peroxygenases from Agrocybeaegerita, Coprinopsis cinerea, Laccaria bicolor and Coprinus radians;also shown as SEQ ID NOs: 1-8 in the present application.

DE 103 32 065 A1 discloses methods for the enzymatic preparation ofacids from alcohols through the intermediary formation of aldehydes byusing the AaP peroxygenase enzyme of Agrocybe aegerita TM A1.

A method was reported for the rapid and selective spectrophotometricdirect detection of aromatic hydroxylation by the AaP peroxygenase(Kluge et al., 2007, Appl. Microbiol. Biotechnol. 75: 1473-1478).

It is well-known that a direct regioselective introduction of oxygenfunctions (oxygenation) into organic molecules constitutes a problem inchemical synthesis. It is particularly difficult to catalyse theselective hydroxylation of aliphatic carbohydrates. The products may beused as important intermediates in a wide variety of differentsyntheses.

In particular the chemical hydroxylation of alkanes is relativelycomplex, requires aggressive/toxic chemicals/catalysts and leads to aseries of undesired by-products.

It is known that an intracellular enzyme, methane monooxygenase (MMO, EC14.13.25), oxygenates/hydroxylates the terminal carbon of somehydrocarbons. The MMO enzyme consists of several protein components andis formed by methylotrophic bacteria (e.g. Methylococcus capsulatus); itrequires complex electron donors such as NADH or NADPH, auxiliaryproteins (flavin reductases, regulator protein) and molecular oxygen(O₂). The natural substrate of MMO is methane, which is oxidized tomethanol. As a particularly unspecific biocatalyst, MMOoxygenates/hydroxylates, as well as methane, a series of furthersubstrates such as n-alkanes and their derivatives, cycloalkanes,aromatics, carbon monoxide and heterocycles. Utilization of the enzymein biotechnology is currently not possible, since it is difficult toisolate, like most intracellular enzymes, it is of low stability, andthe cosubstrates required are relatively expensive.

SUMMARY OF THE INVENTION

In a first aspect, the inventors of the present invention have provideda method for hydroxylation in position 2 or 3 of either end of asubstituted or unsubstituted, linear or branched, aliphatic hydrocarbonhaving at least 3 carbons and having a hydrogen attached to the carbonin position 2 or 3, comprising contacting the aliphatic hydrocarbon withhydrogen peroxide and a polypeptide having peroxygenase activity;wherein the polypeptide comprises:

a) an amino acid sequence which has at least 50% identity to SEQ IDN0:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, or SEQ ID N0:8; and

b) an amino acid sequence represented by one or more of the followingmotifs:

(SEQ ID NO: 9) Motif I: [FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N;(SEQ ID NO: 10) Motif II: G[GN]GX[YF]NXX[VA]AX[EH][LF]R; (SEQ ID NO: 11)Motif III: RXXRI[QE][DEQ]S[IM]ATN; (SEQ ID NO: 12)Motif IV: S[IM]ATN[PG][EQN][FM][SDN][FL]; (SEQ ID NO: 13)Motif V: P[PDK][DG]F[HFW]R[AP]; (SEQ ID NO: 14)Motif VI: [TI]XXXLYPNP[TK][GV].

In an embodiment, the aliphatic hydrocarbon is a fatty acid.

In another aspect is provided a method for hydroxylation in position 2or 3 of the terminal end of an acyl group of a lipid, comprisingcontacting the lipid with hydrogen peroxide and a polypeptide havingperoxygenase activity; wherein the polypeptide comprises:

a) an amino acid sequence which has at least 50% identity to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, or SEQ ID NO:8; and

b) an amino acid sequence represented by one or more of the followingmotifs:

(SEQ ID NO: 9) Motif I: [FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N;(SEQ ID NO: 10) Motif II: G[GN]GX[YF]NXX[VA]AX[EH][LF]R; (SEQ ID NO: 11)Motif III: RXXRI[QE][DEQ]S[IM]ATN; (SEQ ID NO: 12)Motif IV: S[IM]ATN[PG][EQN][FM][SDN][FL]; (SEQ ID NO: 13)Motif V: P[PDK][DG]F[HFW]R[AP]; (SEQ ID NO: 14)Motif VI: [TI]XXXLYPNP[TK][GV].

In further aspects, the invention provides uses of polypeptides havingperoxygenase activity for removal of lipid containing stains fromlaundry; and for reducing unpleasant odor from laundry.

DEFINITIONS

Peroxygenase activity: The term “peroxygenase activity” is definedherein as the capability to hydroxylate naphtalene using hydrogenperoxide, also referred to as “unspecific peroxygenase”, EC 1.11.2.1.This is a heme-thiolate protein. Enzymes of this type includeglycoproteins secreted by agaric basidiomycetes. They catalyse theinsertion of an oxygen atom from H₂O₂ into a wide variety of substrates,including aromatic rings such as naphthalene, toluene, phenanthrene,pyrene and p-nitrophenol, recalcitrant heterocycles such as pyridine,dibenzofuran, various ethers (resulting in O-dealkylation) and alkanessuch as propane, hexane and cyclohexane. Additional reactions which maybe catalysed by peroxygenases include hydroxylation, epoxidation,N-oxidation, sulfooxidation, O- and N-dealkylation, bromination andone-electron oxidations. They have little or no activity towardchloride.

For purposes of the present invention, peroxygenase activity isdetermined according to the spectrophotometric procedure described byKluge et al. (2007, Appl. Microbiol. Biotechnol. 75: 1473-1478).

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the peroxygenase activity of the maturepolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptides of thepresent invention are preferably in a substantially pure form, i.e.,that the polypeptide preparation is essentially free of otherpolypeptide material with which it is natively or recombinantlyassociated. This can be accomplished, for example, by preparing thepolypeptide by well-known recombinant methods or by classicalpurification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having peroxygenase activity that is in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. In a preferred aspect, the mature polypeptide hasthe amino acid sequence shown in positions 1 to 330 of SEQ ID NO:1 basedon the N-terminal peptide sequencing data (Ullrich et al., 2004, Appl.Env. Microbiol. 70(8): 4575-4581), elucidating the start of the matureprotein of AaP peroxygenase enzyme. In another preferred aspect, themature polypeptide has the amino acid sequence shown in positions 1 to328 of SEQ ID NO:2.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277; emboss.org), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the —nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra; emboss.org), preferably version 3.0.0 or later. The optionalparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitutionmatrix. The output of Needle labeled “longest identity” (obtained usingthe —nobrief option) is used as the percent identity and is calculatedas follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment).

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8; or a homologous sequence thereof;as well as genetic manipulation of the DNA encoding such a polypeptide.The modification can be a substitution, a deletion and/or an insertionof one or more (several) amino acids as well as replacements of one ormore (several) amino acid side chains.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having PeroxygenaseActivity (Peroxygenases)

The present invention relates to uses of an isolated polypeptide, whichis preferably recombinantly produced, having peroxygenase activity,which comprises an amino acid sequence having at least 50% identity,preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or98% identity to the polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ IDNO:8.

In a preferred embodiment, the polypeptide comprises an amino acidsequence represented by one or more of the following motifs, preferablycomprising two or more, three or more, four or more, five or six of thefollowing motifs:

(SEQ ID NO: 9) Motif I: [FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N;(SEQ ID NO: 10) Motif II: G[GN]GX[YF]NXX[VA]AX[EH][LF]R; (SEQ ID NO: 11)Motif III: RXXRI[QE][DEQ]S[IM]ATN; (SEQ ID NO: 12)Motif IV: S[IM]ATN[PG][EQN][FM][SDN][FL]; (SEQ ID NO: 13)Motif V: P[PDK][DG]F[HFW]R[AP]; (SEQ ID NO: 14)Motif VI: [TI]XXXLYPNP[TK][GV].

In another embodiment, the polypeptide comprises an amino acid sequencehaving a substitution, deletion, and/or insertion of one or severalamino acids of the mature polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ IDNO:8.

In yet another embodiment, the polypeptide of the first aspect comprisesor consists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ IDNO:8; or a fragment thereof having peroxygenase activity; preferably thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, or SEQ ID NO:8.

Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, AlaNal, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,peroxygenase activity) to identify amino acid residues that are criticalto the activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides that are related to apolypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8is 10, preferably 9, more preferably 8, more preferably 7, morepreferably at most 6, more preferably 5, more preferably 4, even morepreferably 3, most preferably 2, and even most preferably 1.

It is preferable that the polypeptide of the first aspect is encoded bythe polynucleotide contained in the plasmid which is contained in E.coli NN049991 deposited 14 Mar. 2008 under the terms of the BudapestTreaty with the DSMZ under accession number DSM 21289; or which isencoded by the polynucleotide contained in the plasmid which iscontained in E. coli NN049992 deposited 14 Mar. 2008 under the terms ofthe Budapest Treaty with the DSMZ under accession number DSM 21290.

Another preferred embodiment relates to the polypeptide of the firstaspect of the invention, wherein the mature polypeptide is amino acids 1to 330 of SEQ ID NO:1.

Yet another preferred embodiment relates to the polypeptide of the firstaspect of the invention, wherein the mature polypeptide is amino acids 1to 328 of SEQ ID NO:2.

Yet another preferred embodiment relates to the polypeptide of the firstaspect of the invention, wherein the mature polypeptide is amino acids 1to 344 of SEQ ID NO:4.

Hydrogen Peroxide

The hydrogen peroxide required by the peroxygenase may be provided as anaqueous solution of hydrogen peroxide or a hydrogen peroxide precursorfor in situ production of hydrogen peroxide. Any solid entity whichliberates upon dissolution a peroxide which is useable by peroxygenasecan serve as a source of hydrogen peroxide. Compounds which yieldhydrogen peroxide upon dissolution in water or an appropriate aqueousbased medium include but are not limited to metal peroxides,percarbonates, persulphates, perphosphates, peroxyacids, alkyperoxides,acylperoxides, peroxyesters, urea peroxide, perborates andperoxycarboxylic acids or salts thereof.

Another source of hydrogen peroxide is a hydrogen peroxide generatingenzyme system, such as an oxidase together with a substrate for theoxidase. Examples of combinations of oxidase and substrate comprise, butare not limited to, amino acid oxidase (see e.g. U.S. Pat. No.6,248,575) and a suitable amino acid, glucose oxidase (see e.g. WO95/29996) and glucose, lactate oxidase and lactate, galactose oxidase(see e.g. WO 00/50606) and galactose, and aldose oxidase (see e.g. WO99/31990) and a suitable aldose.

By studying EC 1.1.3._, EC 1.2.3._, EC 1.4.3._, and EC 1.5.3._(—) orsimilar classes (under the International Union of Biochemistry), otherexamples of such combinations of oxidases and substrates are easilyrecognized by one skilled in the art.

Hydrogen peroxide or a source of hydrogen peroxide may be added at thebeginning of or during the method of the invention, e.g. as one or moreseparate additions of hydrogen peroxide; or continously as fed-batchaddition. Typical amounts of hydrogen peroxide correspond to levels offrom 0.001 mM to 25 mM, preferably to levels of from 0.005 mM to 5 mM,and particularly to levels of from 0.01 to 1 mM hydrogen peroxide.Hydrogen peroxide may also be used in an amount corresponding to levelsof from 0.1 mM to 25 mM, preferably to levels of from 0.5 mM to 15 mM,more preferably to levels of from 1 mM to 10 mM, and most preferably tolevels of from 2 mM to 8 mM hydrogen peroxide.

Surfactants

The method of the invention may include application of a surfactant (forexample, as part of a detergent formulation or as a wetting agent).Surfactants suitable for being applied may be non-ionic (includingsemi-polar), anionic, cationic and/or zwitterionic; preferably thesurfactant is anionic (such as linear alkylbenzenesulfonate,alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcoholethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methylester, alkyl- or alkenylsuccinic acid or soap) or non-ionic (such asalcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acylN-alkyl derivatives of glucosamine (“glucamides”)), or a mixturethereof.

When included in the method of the invention, the concentration of thesurfactant will usually be from about 0.01% to about 10%, preferablyabout 0.05% to about 5%, and more preferably about 0.1% to about 1% byweight.

Aliphatic Hydrocarbons

The hydrocarbons, which are hydroxylated in the method of the invention,are aliphatic hydrocarbons having a chain of at least 3 carbons, andhaving a hydrogen attached to the carbon in position 2 or 3. Preferably,the aliphatic hydrocarbon is an alkane or an alkene; more preferably,the aliphatic hydrocarbon is an alkane, such as propane, butane,pentane, hexane, heptane, octane, nonane or decane, or isomers thereof.

The aliphatic hydrocarbons are linear or branched, but not cyclic, assite specific hydroxylation is not possible with cyclic hydrocarbons.Branched hydrocarbons correspond to isomers of linear hydrocarbons.

The aliphatic hydrocarbons are substituted or unsubstituted. Preferably,the aliphatic hydrocarbons are unsubstituted, such as non-activatedhydrocarbons.

When the aliphatic hydrocarbons are substituted (functional groupsattached), the preferred substituents are halogen, hydroxyl, carboxyl,amino, nitro, cyano, thiol, sulphonyl, formyl, acetyl, methoxy, ethoxy,phenyl, benzyl, xylyl, carbamoyl and sulfamoyl; more preferredsubstituents are chloro, hydroxyl, carboxyl and sulphonyl; and mostpreferred substituents are chloro and carboxyl.

The aliphatic hydrocarbons may be substituted by up to 10 substituents,up to 8 substituents, up to 6 substituents, up to 4 substituents, up to2 substituents, or by up to one substituent.

In a preferred embodiment, the aliphatic hydrocarbon is a fatty acid(the substituent is a carboxyl group). Examples of fatty acids include,but are not limited to, butanoic acid (butyric acid), pentanoic acid(valeric acid), hexanoic acid (caproic acid), heptanoic acid (enanthicacid), octanoic acid (caprylic acid), nonanoic acid (pelargonic acid),decanoic acid (capric acid), dodecanoic acid (lauric acid),tetradecanoic acid (myristic acid), hexadecanoic acid (palmitic acid),octadecanoic acid (stearic acid), eicosanoic acid (arachidic acid),linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid,and docosahexaenoic acid.

In a second aspect, the aliphatic hydrocarbon is an acyl group of alipid, such as a monoglyceride, diglyceride, triglyceride, phospholipidor sphingolipid; and the hydroxylation takes place in position 2 orposition 3 of the terminal end of the acyl group. The acyl group musthave at least one hydrogen attached to the carbon in position 2 or 3 ofthe terminal end. The acyl group may be saturated or unsaturated, andoptionally functional groups (substituents) may be attached. Examples ofacyl groups include, but are not limited to, the acyl forms of butanoicacid (butyric acid), pentanoic acid (valeric acid), hexanoic acid(caproic acid), heptanoic acid (enanthic acid), octanoic acid (caprylicacid), nonanoic acid (pelargonic acid), decanoic acid (capric acid),dodecanoic acid (lauric acid), tetradecanoic acid (myristic acid),hexadecanoic acid (palmitic acid), octadecanoic acid (stearic acid),eicosanoic acid (arachidic acid), linoleic acid, linolenic acid,arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid.

Methods and Uses

The present invention provides a method for site specific hydroxylationin position 2 or position 3 of an aliphatic hydrocarbon using aperoxygenase and hydrogen peroxide. The aliphatic hydrocarbon mustinclude a chain of at least 3 carbons, and either (one or more) end ofthe aliphatic hydrocarbon may be used as the starting point to determinewhich carbon is in position 2 or 3. The aliphatic hydrocarbon must haveat least one hydrogen attached to the carbon (which is hydroxylated) inposition 2 or 3. In a preferred embodiment, the carbon in position 2 or3, which is hydroxylated with the peroxygenase, is unsubstituted (beforethe hydroxylation is carried out).

Accordingly, in a first aspect, the present invention provides a methodfor hydroxylation in position 2 or 3 of either end (one or more ends) ofa substituted or unsubstituted, linear or branched, aliphatichydrocarbon having at least 3 carbons and having a hydrogen attached tothe carbon in position 2 or 3, comprising contacting the aliphatichydrocarbon with hydrogen peroxide and a polypeptide having peroxygenaseactivity; wherein the polypeptide comprises:

a) an amino acid sequence which has at least 50% identity to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, or SEQ ID NO:8; and

b) an amino acid sequence represented by one or more of the followingmotifs:

(SEQ ID NO: 9) Motif I: [FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N;(SEQ ID NO: 10) Motif II: G[GN]GX[YF]NXX[VA]AX[EH][LF]R; (SEQ ID NO: 11)Motif III: RXXRI[QE][DEQ]S[IM]ATN; (SEQ ID NO: 12)Motif IV: S[IM]ATN[PG][EQN][FM][SDN][FL]; (SEQ ID NO: 13)Motif V: P[PDK][DG]F[HFW]R[AP]; (SEQ ID NO: 14)Motif VI: [TI]XXXLYPNP[TK][GV].

The method of the invention may be used for a variety of purposes, likebulk chemical synthesis (biocatalysis), increasing aqueous solubility ofaliphatic hydrocarbons, bioremediation, and modification of thecharacteristics of food products.

The method of the invention may also be used for a number of industrialprocesses in which said hydroxylation reactions are beneficial. Anexample of such use is in the manufacture of pulp and paper productswhere alkanes and other relevant aliphatic hydrocarbons that are presentin the wood (resin) can result in depositioning problems in the pulp andpaper manufacturing process. These hydrophobic compounds are theprecursors of the so-called pitch deposits within the pulp and papermanufacturing processes. Pitch deposition results in low quality pulp,and can cause the shutdown of pulp mill operations. Specific issuesrelated to pulps with high extractives content include runnabilityproblems, spots and holes in the paper, and sheet breaks. Treatment withperoxygenase can increase the solubility of said compounds and therebymitigate problems.

Yet another use of the method of the invention is in i.e. oil or coalrefineries where the peroxygenase catalyzed hydroxylation can be used tomodify the solubility, viscosity and/or combustion characteristics ofhydrocarbons. Specifically the treatment can lead to changes in thesmoke point, the kindling point, the fire point and the boiling point ofthe hydrocarbons subjected to the treatment.

In the synthesis of bulk chemicals, agro chemicals (incl. pesticides),specialty chemicals and pharmaceuticals the method of the invention mayobviously be relevant in terms of selectively introducing hydroxy groupsin the substrates thereby affecting the solubility of the modifiedcompound. Furthermore, the selective hydroxylation provides a site forfurther modification by methods known in the art of organic chemicalsynthesis and chemo-enzymatic synthesis.

Natural gas is extensively processed to remove higher alkanes.Hydroxylation of such higher alkanes may be used to improve watersolubility, and thus facilitate removal of the higher alkanes by washingthe natural gas stream. Removal may be performed at the well or duringrefining.

Hydroxylation of oil waste will significantly improve biodegradabilityand will be applicable both in connection with waste water treatmentfrom refineries and bioremediation of contaminated ground or water

In a second aspect, the present invention provides a method forhydroxylation in position 2 or 3 of the terminal end of an acyl group ofa lipid, comprising contacting the lipid with hydrogen peroxide and apolypeptide having peroxygenase activity; wherein the polypeptidecomprises:

a) an amino acid sequence which has at least 50% identity to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, or SEQ ID NO:8; and

b) an amino acid sequence represented by one or more of the followingmotifs:

(SEQ ID NO: 9) Motif I: [FL]XX[YF]S[AN]X[FHY]G[GN]GX[YF]N;(SEQ ID NO: 10) Motif II: G[GN]GX[YF]NXX[VA]AX[EH][LF]R; (SEQ ID NO: 11)Motif III: RXXRI[QE][DEQ]S[IM]ATN; (SEQ ID NO: 12)Motif IV: S[IM]ATN[PG][EQN][FM][SDN][FL]; (SEQ ID NO: 13)Motif V: P[PDK][DG]F[HFW]R[AP]; (SEQ ID NO: 14)Motif VI: [TI]XXXLYPNP[TK][GV].

Hydroxylation of the acyl group of a lipid generally improves theaqueous solubility of the lipid. Accordingly, the method of theinvention may be used to remove or reduce oil or lipid containingstains, like chocolate, from laundry, by contacting the laundry with aperoxygenase and a source of hydrogen peroxide, and optionally asurfactant.

The methods of the invention may be carried out with an immobilizedpolypeptide having peroxygenase activity (peroxygenase).

The methods of the invention may be carried out in an aqueous solvent(reaction medium), various alcohols, ethers, other polar or non-polarsolvents, or mixtures thereof. By studying the characteristics of thealiphatic hydrocarbon used in the methods of the invention, suitableexamples of solvents are easily recognized by one skilled in the art. Byraising or lowering the pressure at which the hydroxylation is carriedout, the solvent (reaction medium) and the aliphatic hydrocarbon can bemaintained in a liquid phase at the reaction temperature.

The methods according to the invention may be carried out at atemperature between 0 and 90° C., preferably between 5 and 80° C., morepreferably between 10 and 70° C., even more preferably between 15 and60° C., most preferably between 20 and 50° C., and in particular between20 and 40° C.

The methods of the invention may employ a treatment time of from 10seconds to (at least) 24 hours, preferably from 1 minute to (at least)12 hours, more preferably from 5 minutes to (at least) 6 hours, mostpreferably from 5 minutes to (at least) 3 hours, and in particular from5 minutes to (at least) 1 hour.

In another aspect, the methods of the invention may be used to reduceunpleasant odors from laundry by contacting the laundry with aperoxygenase and a source of hydrogen peroxide, and optionally asurfactant. The method of the invention results in reduction of theamount of butanoic acid (butyric acid) in the laundry. Butanoic acid isformed during washing of laundry when certain animal fats and plant oilsare hydrolyzed, e.g. by detergent lipase, to yield free fatty acids,including butanoic acid. Butanoic acid has an extremely unpleasant odor.The peroxygenase hydroxylates the butanoic acid to 2-hydroxybutyric acid(a/pha-hydroxybutyric acid) or 3-hydroxybutyric acid(beta-hydroxybutyric acid).

Unless otherwise specified, the nomenclature used is standard IUPACnomenclature.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

The polypeptide having peroxygenase activity from Agrocybe aegerita,which is shown as SEQ ID NO:2, is referred to as AaeAPO in the followingexamples.

Example 1 Hydroxylation of N-Hexane

Enzymatic hydroxylation of hexane was performed in the pure substrate(n-hexane, >97%, Sigma Aldrich) containing 2 U ml⁻¹ (0.31 nmol) AaeAPOadded as aqueous enzyme solution (10 μl). H₂O₂ (4 mM) was added bysyringe pumps over 1 hour. The experiment was done in 200 μl scale(total volume) in 1 ml glass vials stirred with a magnetic stirrer.Products were analyzed by GC-MS (Varian) by direct injection of thereaction mixture. Controls were processed identically except that water(10 pl) was added instead of enzyme solution.

The gas chromatogram and mass spectra of the sample with active enzyme(AaeAPO) and n-hexane showed formation of high amounts of 2-hexanol, and3-hexanol; the control without enzyme did not contain any of thesepeaks.

Example 2 Hydroxylation of N-Decane

Enzymatic conversion (in 200 μl total volume) was done in pure n-decane(>97%, Sigma Aldrich) supplemented with 2 U ml⁻¹ (0.31 nmol) AaeAPO.H₂O₂ (8 mM) was added by syringe pumps over 2 hours and the sample wasstirred with a magnetic stirrer. Products were measured by GC-MS.Controls were processed identically except that water (10 μl) was addedinstead of enzyme.

The gas chromatogram and mass spectra of the sample with active enzyme(AaeAPO) and n-decane showed formation of high amounts of twon-alkanols, 3-decanol and 2-decanol; the control without enzyme did notcontain these peaks.

Example 3 Enzymatic Hydroxylation of Lauric Acid

Enzymatic hydroxylation of lauric acid was performed using a totalreaction mixture of 4 ml containing 50 mM potassium phosphate buffer, 40v/v % acetronitrile, 1 mM lauric acid (>98% pure, Aldrich W261408 wasdissolved in acetonitrile), 0.01 mg peroxygenase protein/ml (theperoxygenase shown as SEQ ID NO:4) and 2 mM ascorbic acid was addedaccording to the table below.

The reaction was started by addition of hydrogen peroxide correspondingto a concentration of 0.5 mM in the reaction mixture. The reactionmixtures were incubated for 60 minutes at 35° C. using a heat block. Asecond addition of peroxide was added after 30 minutes incubation to atotal concentration of 1 mM. The reactions were stopped by a heattreatment of 85° C. in a water bath for 5 minutes. Products weremeasured by GC-FID (Varian 3900) by injection at 100° C. in split modewith ratio of 10:1 (helium was used as carrier gas at a constant flow of25 ml/min). A temperature gradient were applied heating to 200° C. at arate of 10° C./min, then proceeding to 360° C. at a rate of 50° C./min.The results were recorded as peak area (see Table 1).

TABLE 1 Lauric acid Product Treatment (Area@3.2 min) (Area @4.3 min)Peroxygenase + H₂O₂ No peak No peak Lauric acid + H₂O₂ + ascorbic acid16011 91 Lauric acid + peroxygenase + 14406 395.5 H₂O₂ + ascorbic acid

A product peak appeared in the presence of the peroxygenase. The elutiontime of the product was slightly shifted compared to12-Hydroxydodecanoic acid, which is an iso-form of 2-hydroxy lauric acidand 3-hydroxy lauric acid. Hence, the elution time was in accordance tothe expected product hydroxylated in the 2 or 3 position.

Example 4 Enzymatic Hydroxylation of Palmitic Acid

Enzymatic hydroxylation of palmitic acid was performed using a totalreaction mixture of 4 ml containing 50 mM potassium phosphate buffer, 40v/v % acetronitrile, 1 mM palmitic acid (>99% pure, Sigma P0500) and0.01 mg peroxygenase protein/ml (the peroxygenase shown as SEQ ID NO:4).

The reaction was started by addition of hydrogen peroxide correspondingto a concentration of 1 mM in the reaction mixture. The reactionmixtures were incubated for 1, 2, 3 and 10 minutes at 35° C. using aheat block. The reactions were stopped by a heat treatment of 85° C. ina water bath for 5 minutes. Products were measured by GC-FID (Varian3900) by injection at 100° C. in split mode with ratio of 10:1 (heliumwas used as carrier gas at a constant flow of 25 ml/min). A temperaturegradient were applied heating to 200° C. at a rate of 10° C./min, thenproceeding to 360° C. at a rate of 50° C./min. The results were recordedas peak area (see Table 2).

TABLE 2 Incubation time Palmitic acid Product (min) (Area) (Area) 01799.5 No peak 1 1481.2 82.8 2 979.1 191.3

A product peak appeared already after 1 minutes of incubation, andincreased after two minutes incubation. The elution profile was slightlyshifted compared to 16-Hydroxyhexadecanoic acid, which is an iso-form of2-hydroxy palmitic and 3-hydroxy palmitic. Hence, the elution time wasin accordance to the expected product hydroxylated in the 2 or 3position.

1-17. (canceled)
 18. A method for hydroxylation in position 2 or 3 ofeither end of a substituted or unsubstituted, linear or branched,aliphatic hydrocarbon having at least 3 carbons and having a hydrogenattached to the carbon in position 2 or 3, comprising contacting thealiphatic hydrocarbon with hydrogen peroxide and a polypeptide havingperoxygenase activity; wherein the polypeptide has at least 90% sequenceidentity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, or SEQ ID NO:8.
 19. The method of claim 18, whereinthe carbon in position 2 or 3, which is hydroxylated, is unsubstituteduntil it is contacted with the peroxygenase.
 20. The method of claim 18,wherein the amino acid sequence of the polypeptide comprises SEQ IDNO:9.
 21. The method of claim 18, wherein the amino acid sequence of thepolypeptide comprises SEQ ID NO:10.
 22. The method of claim 18, whereinthe amino acid sequence of the polypeptide comprises SEQ ID NO:11. 23.The method of claim 18, wherein the amino acid sequence of thepolypeptide comprises SEQ ID NO:12.
 24. The method of claim 18, whereinthe amino acid sequence of the polypeptide comprises SEQ ID NO:13. 25.The method of claim 18, wherein the amino acid sequence of thepolypeptide comprises SEQ ID NO:14.
 26. The method of claim 18, whereinthe polypeptide has at least 95% sequence identity to SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ IDNO:8.
 27. The method of claim 18, wherein the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
 28. Themethod of claim 18, wherein the polypeptide is a fragment thereof havingperoxygenase activity.
 29. The method of claim 18, wherein thesubstituents of the aliphatic hydrocarbon are selected from the groupconsisting of halogen, hydroxyl, carboxyl, amino, nitro, cyano, thiol,sulphonyl, formyl, acetyl, methoxy, ethoxy, phenyl, benzyl, xylyl,carbamoyl and sulfamoyl.
 30. The method of claim 18, wherein thesubstituents are selected from the group consisting of chloro, hydroxyl,carboxyl and sulphonyl; in particular chloro and carboxyl.
 31. Themethod of claim 18, wherein the aliphatic hydrocarbon is unsubstituted.32. The method of claim 18, wherein the aliphatic hydrocarbon is linear.33. The method of claim 18, wherein the aliphatic hydrocarbon is analkane.
 34. The method of claim 33, wherein the alkane is propane,butane, pentane, hexane, heptane, octane, nonane or decane, or isomersthereof.
 35. The method of claim 18, wherein the aliphatic hydrocarbonis part of a fatty acid.
 36. A method for hydroxylation in position 2 or3 of the terminal end of an acyl group of a lipid, comprising contactingthe lipid with hydrogen peroxide and a polypeptide having peroxygenaseactivity; wherein the polypeptide has at least 90% sequence identity toSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, or SEQ ID NO:8.
 37. The method of claim 36, wherein thepolypeptide has at least 95% identity to the mature polypeptide of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, or SEQ ID NO:8.